Trichoderma Reesei Pyrg Gene Mutant Strain Screening And Gnti And Vhb Gene Expression

Filamentous fungus has the advantages of simple structure and fast growth, so it is easy to manipulate and culture. Being a kind of lower eukaryotes, it also has eukaryotic protein modification apparatus and the post translation modification mechanism, which is similar to that of mammals. Filamentous fungi are commonly used in fermentation industry for large scale production of enzmys and organic acids, several of these enzymes can be produced up to 20～40g per liter. So molecular biological research on filamentous fungus has been one of the hotspots. A start has been made to engineer the glycosylation path way in filamentous fungi to obtain stains that show a more mammalian-like type of glycosylation.Trichoderma reesei is an asexually reproducing filamentous fungus, mainly used in cellulase, hemicellualse production. The molecular evidence shows that T. reesei is an asexual, clonal line derivative of the ascomycete Hyporcea jecorina. Trichoderma reesei has the typical eukaryotic character of protein modification as well as the ideal capability of secreting proteins. Recently, T. reesei has been used in production of heterologous eukaryotic proteins as a host stain and genetic modification has been made for the therapeutic proteins production, such as the humanization of N-glycosylation pathways.In order to use the pyrG gene select marker in the transformation of T. reesei, first we obtained a T. reesei pyrG gene deficient strain M23 by using the UV mutation technology. The strain M23 cannot survive on medium without uridine until it is transformated with the vector of the pyrG gene. The transformation frequency is approximate 200～300 transformants per μg plasmids.In this study, the fungal express vector pCGNT used to express the human N-acetylglucosaminyltransferase I was constructed with the promoter and terminator of T. reesei cbh1. After that, the expression cassette of Vitreoscilla Hemoglobin (VHb) contains the promoter and terminator of A. nidulans trpC was inserted into pCGNT to...